Porous scaffolds for dermal tissue engineering were fabricated by freeze-drying a mixture of chitosan and gelatin (CG) solutions. Different crosslinking agents including glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethyl-carbodimide hydrochloride (EDC), and genipin were used to crosslink the scaffolds and improve their biostability. The porous structure and mechanical properties were determined for the scaffolds. The proliferation of human fibroblasts in the scaffolds was analyzed. It was found that EDC crosslinked scaffolds had the greatest amount of cells after four days. EDC crosslinked CG scaffolds had tensile modulus in a dry state and compressive modulus in a wet state similar to commercial collagen wound dressing. They also showed appropriate pore size, high water absorption, and good dimensional stability during cell culture. When human fibroblasts were seeded on acellular porcine dermis (APD), acellular human dermis (AHD), and CG scaffolds for 3D cell culture, they were well-distributed in the centre of the CG scaffolds but stayed only on the superficial layer of APD or AHD after seven days. A gelatin-based bioglue was applied to the CG scaffolds where the keratinocytes were seeded to mimic epidermal structure. After 14 days, the bioglue degraded and keratinocytes grew to form monolayers on the scaffolds. This study showed that CG scaffolds crosslinked by EDC and seeded with human fibroblasts could serve as dermal constructs, while the bioglue coating seeded with keratinocytes could serve as an epidermal construct. Such a combination could help regenerate skin with integrated dermal and epidermal layers and a have potential use in tissue-engineered skin.

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Rheumatoid arthritis (RA) is a systemic autoimmune disease affecting 1% of the world’s population, with significant morbidity and mortality. In this study, we investigated a recombinant adeno-associated virus (rAAV) vector for its potential application in RA gene therapy. rAAV encoding Escherichia coliβ-galactosidase was injected into rat joints which had already been induced into acute arthritis after local lipopolysaccharide (LPS) administration, and the efficiency of in vivo transduction was evaluated. We observed a striking correlation between vector transgene expression and disease severity in arthritic joints. The inflammatory reaction peaked at 3 to 7 days after LPS treatment, and, at the same time, 95% of the synoviocytes had high-level transgene expression. Gene expression diminished to the basal level (5%) when the inflammation subsided at 30 days after LPS treatment. More importantly, the diminished transgene expression could be efficiently reactivated by a repeated insult. The transgene expression in normal joints transduced with rAAV remained low for a long period of time (30 days) but could still be induced to high levels (95%) at 3 to 7 days after LPS treatment. This is the first demonstration of disease state-regulated transgene expression. These findings strongly support the feasibility of therapeutic as well as preventative gene transfer approaches for RA with rAAV vectors containing therapeutic genes, which are expected to respond primarily to the disease state of the target tissue.

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